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Screening of Corticosteroids in Urine by Negative Atmospheric Pressure Chemical Ionization LC/MS/MS

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α-glucuronidase from E. Coli K12, from Roche Diagnostics, Germany

All other chemicals are pro analysis or HPLC grade

Sample Preparation

The samples are prepared by a standard procedure for steroids. A 2 mL urine sample is transferred in a tube. A 40 μL aliquot of a 10 ppm desoximetasone (internal standard) solution, 1 mL phosphate buffer 0.8M pH 7.0 and 25 μL beta-glucuronidase are added, the mixture is vigorously vortexed and kept for 1 hour at 50 C for enzymatic hydrolysis. (The enzymatic hydrolysis step is needed since the corticosteroids are mainly excreted in a conjugated form with the glucuronic acid). Adding 750 μL of 20% buffer K2CO3/ KHCO3 (1:1), which brings the pH around 9, stops the hydrolysis. Next, 5 mL of tertbutylmethylether is added, and the mixture shaken for 15 min. After centrifugation, the organic layer is transferred to another tube and evaporated to dryness. The remaining residue is dissolved in 100 μL mobile phase (20:80, solvent A / solvent B). Then, 10 μL is injected in LC/MS/MS [5].

Results and Discussion

In order to develop the MS parameters, 10 ppm solutions of each corticosteroid were prepared in a 20% buffer A / 80% methanol mixture. The mixture was meant to mimic the mobile phase that would elute with the compound of interest in an actual LC/MS analysis. The 10 ppm solutions were directly injected in the APCI with a syringe pump at a 50 μL/min rate. First, the most appropriate precursor ion was selected from the parent scan, and the capillary voltage was optimized for its highest abundance. Second, the product ions were selected and the collision energies optimized by the MS/ MS breakdown automatic procedure.

Two MS/MS product ions, instead of one, are used to monitor each corticosteroid in ord
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